The suspected relationship between neoplastic disease and thromboembolic disorders, first recognized by Armand Trousseau in 1865, who described an increased incidence of venous thrombosis in a series of patients with gastric carcinoma, led to the identification of abnormal fibrin metabolism with malignant disease. An increased rate of fibrinogen turnover (Yoda et al. (1981) Thromb. Haemost. 46:706-709), an increase in plasma levels of fibrinogen/fibrin-related antigen (Merskey et al. (1980) Br. J. Haematol 44:655-670), and an increase in plasma levels of fibrinopeptide-A (a 16-amino acid peptide cleaved from the A chain of fibrinogen by thrombin) have been observed in virtually all patients with acute leukemia and solid tumors (Yoda et al. (1981) supra). It is believed that subclinical activation of blood coagulation may be a reflection of the interdependence of tumor growth and fibrin generation (Rickles et al. (1983) Blood 62:14-31). Fibrin was found deposited on the advancing margin of solid tumors and also on blood-borne, potentially metastatic, malignant cells. Administration of anticoagulants and fibrinolysins decreased tumor growth and metastasis (Wood et al. (1955) in The Pathogenesis of Cancer, J. E. Gregory (ed.), Fremont Foundation, Pasadena, Calif., pp. 140-151). Thus, it appeared clear that altered fibrin metabolism was associated with malignant tissue, but it was not clear what caused the altered activity of the coagulation cascade that resulted in abnormal fibrin deposition.
To explain the hypercoagulable state associated with neoplasia, the concept emerged that malignant tissue produced a substance capable of initiating coagulation. Among the mechanisms that can promote abnormal activation of blood clotting in malignancy, tumor-associated procoagulants are considered to play a prominent role. In 1975 Gordon et al. (Thromb. Res. 6:127-137) reported the isolation of a protein called cancer procoagulant (CP) from rabbit V2 carcinoma cells that initiated coagulation by a mechanism that was distinguishable from that of the intrinsic and extrinsic pathways. CP was shown to be different from tissue factor, which is normally released from damaged cells and participates via the extrinsic pathway in the activation of the coagulation system. It was shown that CP initiated coagulation in the absence of factor VII and was inhibited by diisopropylfluorophosphate (DFP), two characteristics that distinguish CP from tissue factor. CP has now been characterized as a cysteine proteinase having a molecular weight of 68,000 and capable of initiating coagulation by directly activating factor X in the coagulation cascade (Gordon, U.S. Pat. No. 4,461,833; Falanga et al. (1985) Biochem. 24:5558-5567, and Biochim. Biophys. Acta 831:161-165). Most importantly, although CP could be extracted from tumor cells, no CP activity or antigen could be detected in extracts from normal cells or from benign melanocytic lesions (Donati et al. (1986) Cancer Res. 46:6471-6474; Falanga et al. (1988) Blood 71:870-875). The presence of CP was clearly associated with the malignant phenotype and its activity appears to be particularly high in metastatic cells.
For years investigators had sought to identify diagnostic markers of cancerous cells for the purpose of developing cancer therapies. In 1970, Bubenek et al. (Int. J. Cancer 5:310), demonstrated that serum from cancer patients contained antibodies that bound to tumor cell surface antigens Subsequently, many reports were published on the presence of antigens on the surface of human melanoma and on other neoplastic cells. The ultimate goal in these investigations was to utilize the corresponding specific antibodies in the immunotherapy of cancer.
Of the many antigens characterizing tumor cells, some of the more notable are carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP) and acute lymphoblastic leukemia associated antigen (cALLA) A number of other tumor-associated antigens have been studied as possible tumor detectors (Hellstrom et al. (1982) Springer Semin. Immunopathol. 5:127). In most cases there is little evidence to indicate that any of these antigens have value as a potential tumor marker.
Investigation was also undertaken to test different drug therapies for their effectiveness in fighting neoplasia. In 1976 the hypothesis was tested in man that anticoagulant drugs could modify favorably the course of human cancer. Warfarin, an agent shown to be effective in a variety of animal tumor models, was found to be therapeutically associated with (i) a significant prolongation in the time to first evidence disease progression and, (ii) a significant improvement in survival for patients with small cell carcinoma of the lung (Zacharski et al. (1984) Cancer 53:2046). In 1981, the antiplatelet drug RA-233 (mopidamol) was tested for treatment of human malignancy. RA-233 is a phosphodiesterase inhibitor that has been shown to limit progression of tumors in experimental animal models (Zacharski et al. (1982) Am. J. Clin. Oncol. 5:593). In this study, RA-233 was associated with a statistically significant prolongation of survival in patients with nonsmall cell lung cancer limited to one hemithorax (Zacharski et al. (1987) Thromb. Haemost. (Suppl) 58:508). None of the other tumor types was found to respond to treatment with RA-233. Although it is possible that both of these drugs, warfarin and RA-233, may act outside the role of anti-coagulant/platelet agent, this approach was considered to have merit and is under further examination for continuation.
It is still believed today that the finding of a selective marker diagnostic of tumorigenic cells would enable detection of malignant cells and would then allow treatment of neoplasia. Such a diagnostic marker was shown to be CP, first isolated from rabbit V.sub.2 carcinoma (Gordon et al. (1975) Thromb. Res. 6:127-137; (1981) J. Clin. Invest. 67:1665-1671). CP, a cysteine proteinase, was subsequently found in tissue extracts of the following human cancers: breast, colon, vagina, kidney, lung, blood and bone marrow (Gordon et al. (1975) supra; (1979) J. Natl. cancer Inst. 62:773-776; Falanga et al. (1988) Blood 71:870-875). CP has also been described in transformed hamster fibroblasts (Gordon et (1978) Cancer Res. 38:2467-2472), mouse Lewis lung carcinoma (3LL) cells (Colucci et al. (1980) Thromb. Res. 18:589-595), B16 mouse melanoma cells (Donati et al. (1986) Cancer Res. 46:6471), mouse Ehrlich ascites carcinoma cells (Curatolo et al. (1979) Br. J. Cancer 40:228-233), and mouse JW sarcoma cells (Curatolo et al. (1979) supra). However, in none of these studies was CP found in extracts of adjacent normal tissue. Thus, CP appeared to be a diagnostic marker associated exclusively with the malignant state.
Cysteine proteinases constitute a major class of proteolytic enzymes characterized by an active sulfhydryl group that participates in catalysis by means of acylthioester formation (Fink et al. (1976) Biochemistry 15:5287-5293). Proteinases of this class have been shown to function in protein turnover (Dayton et al. (1976) Biochemistry 15:2150-2158), hormone metabolism (Rupnow et al. (1979) Biochemistry 18:1206-1212) and viral protein processing (Korant et al. (1979) Proc. Natl. Acad. Sci. 76 2992-2995). In order to be able to eventually study the in vivo activities of individual cysteine proteinases, selective inhibitors for enzymes of this class have been prepared and examined first in in vitro studies.
Compounds having the reactive group, diazomethyl ketone, were the first to be examined as potential reagents selective for cysteine proteinases. Carbobenzyloxyphenylalanyl diazomethyl ketone (Z-Phe diazomethyl ketone) and carbobenzyloxy-phenylalanylphenylalanyl diazomethyl ketone (Z-Phe-Phe diazomethyl ketone) were found to be irreversible inhibitors of papain, a model cysteine proteinase from papaya (Leary et al. (1977) Biochemistry 16:5857-5861). The dipeptide derivative was about 200-fold more effective an inhibitor than Z-Phe diazomethyl ketone. Moreover, more importantly, these diazomethyl ketones were found to be without effect on serine proteinases (Leary et al. (1977) supra), metalloproteinases and their effect on aspartate proteinases requires copper ions (Shaw et al. (1980) Biochem. J. 186:385-390). Because of this relative chemical inertness, peptide diazomethyl ketones were deemed to be useful for the study of the role of sulfhydryl proteinases in normal and pathological processes.
Both Z-Phe diazomethyl ketone and Z-Phe-Phe diazomethyl ketone were shown to also inhibit cathepsin B. The dipeptide derivative was three orders of magnitude more effective, indicating the importance of occupation of an additional binding site (Leary et al. (1977) Biochem. Biophys. Res. Comm. 79:926-931). Cathepsin B, a lysosomal cysteine proteinase present in many animal tissues, plays a regulatory role in protein turnover. In addition, cathepsin B has been shown (Burleigh et al. (1974) Biochem. J. 137:387-398) to possess collagenolytic activity, which may be responsible for initial stages of extracellular breakdown of connective tissue (Holtzman (1975) Lysosomes: A Survery, Springer-Verlag, New York, pp. 110-115 and 179-184). Again, the availability of enzyme inhibitors selective for only cysteine proteinases would permit exploration of the potential role of cysteine proteinases, e.g., cathepsin, in metastasis and in other metabolic processes.
A third inhibitor of this class, Z-Phe-Ala diazomethyl ketone, was shown to be a powerful inactivator of bovine spleen cathepsin B with K.sub.i =1.7-10.sup.-6 M (Watanabe et al. (1979) Biochem. Biophys. Res. Comm. 89:1354-1360). The idea that the rate of reaction of a cysteine proteinase with various inhibitors depended on the specificity of the enzyme was tested on cathepsin B and L, both cysteine proteinases of lysosomal origin. The role of cathepsin L is probably not limited to lysosomal protein degradation since precursor forms are secreted by fibroblasts upon transformation and by stimulated inflammatory macrophages (Kirschke et al. (1988) FEBS Lett. 228:128-130). Under assay conditions comprising a very sensitive substrate, Z-Phe-Arg-4-methyl-7-coumarylamide, Z-Phe-Ala dimethyl ketone exhibited an affinity for cathepsin L which was about 2000-fold higher than for cathepsin B from rat and human. Z-Phe-Phe diazomethyl ketone, on the other hand, selectively inactivated cathepsin L in a certain concentration range, whereas it reacted reversibly with cathepsin B from several species (Kirsche et al. (1981) Biochem. Biophys. Res. Comm. 101:454-458).
In 1981 Green and Shaw (J. Biol. Chem. 256:1923-1928) undertook a comprehensive examination of a number of different diazomethyl ketones and compared their rates of inactivation of the endopeptidases clostripain, an enzyme of trypsin-like specificity, streptococcal proteinase and cathepsin B and an exopeptidase, cathepsin C. A number of interesting features emerged: (i) Z-Phe-Ala diazomethyl ketone, one of the most effective inactivators of cathepsin B and L, was absolutely inert toward clostripain, illustrating the type of selectivity attainable by this approach; (ii) the charged amino acid derivative, Z-Lys diazomethyl ketone, proved to be a powerful inhibitor of cathepsin B; (iii) cathepsin B and the streptococcal proteinase were particularly sensitive to reagents having phenylalanine in the penultimate, or P.sub.2, position, rather than in P.sub.1 ; (iv) for cathepsin B, variation in P.sub.1 in the series Z-Gly-Gly-Y diazomethyl ketone does not yield an effective inhibitor, reinforcing the belief that phenylalanine in P.sub.2 contributes greatly to binding. In later studies, Kirschke et al. (1988) FEBS Lett. 228:128-130 found that in contrast to cathepsin B the P.sub.1 region of cathepsin L has the ability to accommodate large hydrophobic side chains, and they showed that Z-Phe-Tyr(O-t-Bu) diazomethyl ketone inactivated cathepsin L with a rate 2.5.times.10.sup.4 greater than that for cathepsin B.
Overall, these results demonstrated that the effectiveness of the inhibitors correlated with the specificities of the individual enzymes. Furthermore, it was shown that peptidyl diazomethyl ketones are unreactive with thiols, such as mercaptoethanol, mercaptoethylamine and glutathione, and with nonproteinase enzymes having an active center thiol. This property and the lack of reactivity toward other classes of proteinases suggest the usefulness of this class of peptidyl inhibitors as diagnostic reagents for both in vitro and in vivo studies (Green et al. (1981) supra).
A different group of peptide derivatives containing a sulfonium salt was found by Shaw (1988) J. Biol. Chem. 263;2768-2772, to act as affinity-labelling reagents of cysteine proteinases. Peptides containing hydrophobic side chains, e.g., Z-He-Ala-CH.sub.2 S.sup.+ (CH.sub.3).sub.2 showed considerable effectiveness for the inactivation of papain and cathepsin. Sulfonium salts having the general structure Z-Lys-CH.sub.2 S.sup.+ -(alkyl).sub.2 irreversibly inactivated the cysteine proteinase clostripain, but also appeared to inhibit two serine proteinases, although with complex kinetic responses. The affinity of the peptidyl sulfonium salts tested was in the nanomolar range for clostripain, whereas for those serine proteinases showing an effect, the affinities were approximately 10 .mu.M (Rauber et al. (1988) Biochem. J. 250:871-876). It is believed that peptidyl methylsulfonium salts are effective as cysteine proteinase inhibitors and that they can be used to study selected enzymes.
The discovery that certain peptidyl diazomethyl ketones and peptidyl sulfonium salts were selective in vitro inhibitors of cysteine proteinases raised the possibility that these inhibitor compounds would be used in vivo to probe the role of different cysteine proteinases in various metabolic processes. Thus, the overall effect of these reagents on protein metabolism was tested in isolated rat hepatocytes. Z-Phe-Phe diazomethyl ketone and Z-Phe-Ala diazomethyl ketone were found (Grinde (1983) Biochim. Biophys. Acta 7575:15-20)to reduce lysosomal protein degradation and synthesis. At a concentration of 10.sup.-4 M, both diazomethyl ketones caused between 55 and 70% inhibition of protein degradation and between 45 and 65% inhibition of protein synthesis. At lower concentrations, e.g., 10.sup.-5 M, Z-Phe-Al a diazomethyl ketone inhibited protein synthesis by 21% whereas Z-Phe-Phe diazomethyl ketone showed 16% inhibition. The substantial suppression of protein metabolism by these two peptidyl diazomethyl ketones indicates that particular caution will have to be exercised in monitoring possible side reactions associated with the utilization of these inhibitory reagents in in vivo or on cellular systems.
Since CP, a cysteine proteinase, appears to be a diagnostic marker of neoplasia, the identification of compounds that act as inhibitors of CP would permit detailed investigation of the role played by CP in malignancy and also of the association between thromboembolic disorders and neoplastic disease. To date, chemical compounds that show specific inactivation of CP have not been reported.